Electrophoretic techniques in element species analysis. Comparison between flow-through and flat-bed techniques

A comparison of electrophoretic techniques for the separation of metal-protein complexes from food extracts is described. A preparative flow-through electrophoresis system with continuous elution of the fractionated substances and an analytical flat-bed gel electrophoresis technique with off-line electro-elution are optimized with regard to this separation problem. The metal-protein complexes are extracted from four flour samples using Tris-glycine buffer (pH 8.3). For the separation, polyacrylamide gels of 14% T and 3% C are prepared and Tris-HCl (pH 8.9) is used as an electrophoresis buffer. For a soy bean flour, not only the separation of protein fractions is achieved but also the metal distribution patterns that are determined by flame atomic absorption spectrometry are given. The results show that the use of the flow-through technique is limited to special fields of application, whereas the flat-bed electrophoresis with subsequent electro-elution of metal-protein complexes is a useful technique in element species analysis.     

Experimental studies on affinity chromatography in an electric field.

A multicompartment electrolyzer, which has been used for preparative electrophoresis [Z. Liu, Z. Huang, J.-Y. Cong, et al., Sep. Sci. Technol. 31 (1996) 427], is applied for carrying out affinity chromatography in an alternating electric field. The effect of electric field strength on the adsorption and desorption characteristics is experimentally examined with human serum albumin and Blue Sepharose Fast Flow as a model system. It is shown that the existence of an electric field leads to a significant change in the adsorption capacity of the blue dye, which may be used for establishing a preferential adsorption to achieve a high resolution. The adsorption speed increases slightly with respect to the increase of electric field strength, while adsorption capacity in the presence of an electric field is independent of the electric field strength. Different elution behavior is observed as function of adsorption condition and a high recovery of the adsorbed protein is obtained when the adsorption is carried out in the presence of an electric field.     

Electrophoretic analysis of phospho and non-phospho protein mixtures extracted from zebrafish whole brain samples by immobilized metal affinity electrophoresis

Phosphoproteins are principle cellular protein components with diverse regulatory functions and phosphorylation is the most frequent post-translational modifications of proteins. Immobilized metal affinity electrophoresis (IMAEP) is a recently developed technique by which the phosphoprotein components of the cellular samples could be captured. We have made use of this new methodology to capture the whole phosphoproteins of zebrafish brain. Since the elution and resolution of captured phosphoproteins by this new methodology are not yet quite developed, we have tried to make this new methodology more efficient in (1) capturing phosphoproteins from biological samples and (2) elution and resolution of captured phosphoproteins. In this project, we first examined the captured phosphoproteins from zebrafish whole brain samples, as a mixture of phosphoproteins and non-phosphoproteins, examined and resolved the captured phosphoproteins by electrophoresis, and finally eluted them successfully from the gel. In this work, we provided an efficient methodology for the elution of captured phosphoproteins from the gel which is an important development in IMAEP in the analysis of phosphoprotein component of cellular samples and showed the possibility of elution of the captured phosphoproteins. The developed methodology will potentially have wide applications in profiling phosphoproteins from biological samples like zebrafish brain and also in studies about signal transduction systems.     

A multichannel gel electrophoresis and continuous fraction collection apparatus for high‐throughput protein separation and characterization

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A “Counter Free‐Flow” elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high‐resolution separation of a complex protein mixture can be achieved on this system using SDS‐PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48–96 fractions over a mass range of ～10–150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 μL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 μg per channel and reduced resolution.     

Unlimited increase in the resolution of DNA ladders

Fractionation of DNA ladders by gel electrophoresis is limited by the progressive compressing of the long DNA end of a ladder. Improvement in the resolution of this DNA is achieved by use of the following two-step electrophoresis. Initially, the DNA ladder is fractionated by conventional constant field agarose gel electrophoresis. Subsequently, gel electrophoresis is performed in the reverse direction by pulsing the electrical field (PFGE). A newly developed type of pulsing is used, which causes inversion of a double-stranded DNA ladder: the distance migrated increases as the length of the DNA molecule increases. Thus, the resolution of DNA bands continues to increase during the PFGE. These two stages of electrophoresis are serially repeated. Eventually, both the short and the long DNA ends of the ladder migrate out of the gel while a selected region of the ladder undergoes progressive increase in resolution during back-and-forth migration. Improved resolution of DNA bands is achieved, without a known limit.     

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies.

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14-66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 +/- 2.3%. A membrane-bound protein, rat skeletal muscle Ca(2+)-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.     

Nonlinear electrophoresis of protein-detergent complexes

A comparatively new procedure is described for the nonlinear electrophoresis of proteins. Movement and separation of complexes formed by proteins and ionic detergents is first experimentally demonstrated for SDS rainbow colored protein molecular weight markers (Amersham). This result was revealed by SDS-PAGE in an asymmetric zero average pulsed electric field with a peak amplitude of up to 300 V cm(-1) and a frequency of 100 Hz. The highest molecular weight fractions were found to have the highest nonlinear drift velocity. A two-dimensional map of distribution of the protein complexes developed using nonlinear electrophoresis followed by SDS gel electrophoresis in an orthogonal direction, reveals nonuniform distribution of the fractions. Nonlinear electrophoresis can be run without electrode chambers, since the buffer electrolyte is not used up in alternating electric fields. Thus, this new type of electrophoresis can have advantages in microfluidic systems and biochips. Also possible uses are discussed of nonlinear electrophoresis via nonlinear focusing of protein-detergent complexes for further improvement of the SDS-PAGE technique for the separation and examination of these large hydrophobic complexes.     

Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis

We report a subdivision of the mitochondrial proteome into defined sets of proteins, which is based on the combination of three different gel electrophoresis procedures. First, Blue-native polyacrylamide gel electrophoresis is employed to separate mitochondrial protein complexes. The protein complexes are electroeluted and completely detached from Coomasssie blue. Subsequently the subunits of the protein complexes are separated by isoelectric focusing and finally by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The resolution capacity of the procedure is demonstrated for the ATP synthase complex, the cytochrome c reductase complex and the preprotein translocase of the outer mitochondrial membrane (the TOM complex). The method allows the separation of isoforms of subunits forming part of protein complexes, whose occurrence seems to be rather a rule than an exception in higher eukaryotes. Furthermore, extremely hydrophobic proteins are detectable on the gels.     

电泳技术在纳米颗粒分离中的应用

合成纳米颗粒常在尺寸和形状方面具有广泛分布.在很多实验中,需要利用一定大小及形状的纳米颗粒的独特物理化学性质,因此,简便快速的纳米颗粒分离技术越来越受到诸多科学领域的重视.电泳技术以其高分辨率,被广泛用于多种生物大分子如核酸、蛋白质等的分离纯化.纳米颗粒在尺寸上与生物体中的蛋白复合物、细胞器和微生物等十分接近,考虑到带电纳米颗粒与生物分子在电场中的运动行为的相似性,运用电泳技术进行纳米颗粒的鉴定、分离和纯化是一种新的思路,并取得了良好的实验结果.本文主要介绍了琼脂糖凝胶电泳、毛细管电泳以及其他一些电泳技术在纳米颗粒分离中的研究进展.     

基于电迁移的蛋白质制备技术和方法新进展

基于电迁移的蛋白质制备技术是对一类分离和制备技术的统称,其特征是在电场的作用下对目标物质进行分离和纯化制备,这种技术在生物大分子和蛋白质组的研究中应用广泛。基于电迁移的制备技术主要包括制备型电泳、制备型电色谱、制备型等电聚焦和自由流电泳等。本文对每种制备型电迁移装置的设计、特点和基于该种装置的各种应用方法的优缺点进行了详细阐述,并列举了一些实例。另外,微量级制备型电泳因分离度高、回收率高以及高效快速的优点,在微量级生物样本分析中发挥着日益重要的作用,近年来备受关注,本文也着重关注了这方面的进展,并对基于电迁移的制备技术做了展望。     

Development of new analytical methods for selenium speciation in selenium-enriched yeast material

A sequential extraction allowing the discrimination of water-soluble and non-soluble selenium fractions has been developed to evaluate the availability of selenium (Se) in an Se-enriched yeast candidate reference material. The fractionation of selenium-containing compounds in the extracts was achieved on preparative grade 200 Superdex 75 and columns. It showed that water-soluble selenium is present in several fractions with a large mass distribution. Low-molecular- (< or = 10,000) and high-molecular-mass selenocompounds (range 10,000-100,000) were considered separately for further experiments. The analytical approach for low-molecular-mass selenocompounds was based onanion-exchange HPLC with on-line inductively coupled plasma (ICP) MS for quantitative analysis. Selenocystine, selenomethionine, selenite and selenate were quantified in the fractions isolated in preparative chromatography. The study revealed the existence of various unidentified Se species in yeast material. The Se-containing proteins in the yeast material have been further separated and selenium quantified by the combination of gel electrophoresis and electrothermal vaporization-ICP-MS. This new approach allows the separation of the proteins with high resolution by sodium dodecylsulfate-polyacrylamide gel electrophoresis and the sensitive determination of selenium in the protein bands.     

Multistep purification of an antifreeze protein from Ammopiptanthus mongolicus by chromatographic and electrophoretic methods

Antifreeze proteins (AFPs) are known as thermal hysteresis proteins, which can depress the freezing points of the solution by noncolligative effects, but do not affect the melting points. Although some AFPs have been found in some plants, the identity of most proteins remains unclear, owing to insufficient quantity and quality to characterize them. In this report, we describe the purification of an AFP from the winter leaves of Ammopiptanthus mongolicus using a combination of column chromatography and gel electrophoresis. After homogenization in ascorbate-acid-containing Tris buffers (pH 7.4) the soluble proteins are captured by (diethylamino)ethyl-cellulose 52 material. An elution with 0.1-0.3M KCl leads to a crude active fraction. The crude fraction is further purified on a Superdex 75 prep-grade column and finally a Poros 20HP2 column. A complex, consisting of two proteins with relative molecular masses of 34,700 and 37,100, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, is obtained by this protein purification protocol. The recovery of two proteins from the gel is carried out by electrophoresis. The purified protein, with a molecular mass of 37,100, shows thermal hysteresis activity (THA) and can modify the normal growth of ice crystals. The THA of this purified antifreeze protein is 0.24 degrees C at the concentration of 5 mg/mL.     

Analytical fractionation of microsomal cytochrome P-450 isoenzymes from rat liver by high-performance ion-exchange chromatography

Ion-exchange Fast Protein Liquid Chromatography (FPLC) on Mono Q and Mono S was optimized for the analytical separation of microsomal cytochrome P-450 species from rat liver. The effects of detergent, pH, gradient profile and column load on resolution are demonstrated. Successive application of anion- and cation-exchange chromatography leads to eleven separated P-450 fractions. The altered microsomal P450 pattern after treatment of rats with various inducers is reflected by distinct elution profiles. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzymatic analysis imply that several FPLC fractions contain more than one P-450 species. Preliminary results are presented showing the suitability of immobilized metal affinity chromatography (MAC) for general P-450 fractionation and thus for the further resolution of Mono Q and Mono S fractions. Scale-up for preparative P-450 fractionation is easily done by adapting the optimized analytical FPLC procedures to Q- and S-Sepharose Fast Flow.     

Preparative electrochromatography of proteins in various types of porous media

The performance of commercial and enzymatically modified size-exclusion (SE) gels in electrochromatography was compared for preparative protein separations. Dextran and agarose-based SE gels were subjected to enzymatic digestion under mild conditions. This treatment partially hydrolyzed the gel matrix modifying its pore size distribution. Enzymatic treatment of agarose-based SE gels was found to increase the resolution of the separation. Successful separation of preparative amounts of the A and B forms of beta-lactoglobulin (difference in electrophoretic mobility of 8.5%) was achieved with a high degree of purity using agarose-based SE gels. The four major whey proteins, beta-lactoglobulin, alpha-lactalbumin, BSA and immunoglobulins, were purified from an acid whey preparation. The degree of retention of a protein in electrochromatography followed their free-solution electrophoretic mobility (mu) when the protein was able to enter the gel pores and the ratio of diffusion/mu when the protein was excluded.     

A novel subfractionation approach for mitochondrial proteins: a three-dimensional mitochondrial proteome map

As mitochondria play critical roles in both cell life and cell death, there is great interest in obtaining a human mitochondrial proteome map. Such a map could potentially be useful in diagnosing diseases, identifying targets for drug therapy, and in screening for unwanted drug side effects. In this paper, we present a novel approach to obtaining a human mitochondrial proteome map that combines sucrose gradient centrifugation with standard two-dimensional gel electrophoresis. The resulting three-dimensional separation of proteins allows us to address some of the problems encountered during previous attempts to obtain mitochondrial proteome maps such as resolution of proteins and solubility of hydrophobic proteins during isoelectric focusing. In addition, we show that this new approach provides functional information about protein complexes within the organelle that is not obtained with two-dimensional gel electrophoresis of whole mitochondria.     

Desorption of plasmid DNA from anion exchangers: Salt concentration at elution is independent of plasmid size and load

Detailed studies on the sorption behavior of plasmids on anion exchangers are rare compared to proteins. In this study, we systematically compare the elution behavior of plasmid DNA on three common anion exchange resins using linear gradient and isocratic elution experiments. Two plasmids of different lengths, 8 and 20 kbp, were studied and their elution characteristics were compared to a green fluorescent protein. Using established methods for determining retention characteristics of biomolecules in ion exchange chromatography lead to remarkable results. In contrast to the green fluorescent protein, plasmid DNA consistently elutes at one characteristic salt concentration in linear gradient elution. This salt concentration was the same independent of plasmid size but differed slightly for different resins. The behavior is consistent also at preparative loadings of plasmid DNA. Thus, only a single linear gradient elution experiment is sufficient to design elution in a process scale capture step. At isocratic elution conditions, plasmid DNA elutes only above this characteristic concentration. Even at slightly lower concentrations most plasmids remain tightly bound. We hypothesize, that the desorption is accompanied by a conformational change leading to a reduced number of available negative charges for binding. This explanation is supported by structural analysis before and after elution.     

Separation of proteins of nuclear ribonucleoprotein particles by high-performance liquid chromatography

High-performance liquid chromatography (h.p.l.c.) is used to fractionate the proteins of the 30–40 S nuclear ribonucleoprotein particles. The major core proteins of the particles are eluted from a SynChropak AX-300 anion-exchange column before the more acidic higher-molecular-weight minor particle proteins. Each of the three major core proteins which can be separated from the other particle proteins by preparative polyacrylamide gel electrophoresis are eluted from the SynChropak AX-300 column as one peak. Isoelectric focusing separates each of these three apparently homogeneous peaks into a series of charge isomers ranging in isoelectric pH (pI) from 5.5 to 9.0. The core proteins of the ribonucleoprotein particles have a strong affinity to each other and form aggregates. The elution of each of the charge isomers of the three major proteins in one peak and their elution from an anion exchanger before the elution of the more acidic higher-molecular-weight minor proteins of the nuclear ribonucleoprotein particles is explained by the formation of these aggregates. The separation of the total proteins of the nuclear ribonucleoprotein particles by h.p.l.c. is similar to the separation which can be obtained by preparative electrophoresis but the l.c. technique is simpler, substantially quicker, and adaptable to large-scale preparation.     

Electronic gel protein transfer and identification using matrix-assisted laser desorption/ionization-mass spectrometry

An electronic protein transfer technique is described for achieving the rapid and efficient recovery of sodium dodecyl sulfate (SDS)-protein complexes from polyacrylamide gels. This process involves the use of small-dimension capillaries in physical contact with a resolved protein band within the polyacrylamide gel, providing a large potential drop and high electric field strength at the capillary/gel interface. Several factors controlling the electronic protein transfer, including the applied electric field strength, the electrophoresis buffer concentration, and the capillary dimension, are studied to further enhance the use of field-amplification for sample stacking of extracted SDS-protein complexes. As a result of sample stacking, the extracted proteins from a 50 ng gel loading are present in a narrow ( approximately 80 nL) and highly concentrated (0.46 mg/mL or 3.3 x 10(-5) M for cytochrome c) solution plug. Three model proteins with molecular mass ranging from 14 kDa (cytochrome c) to 116 kDa (beta-galactosidase) are stained by Coomassie blue and electrophoretically extracted from gels with protein loadings as low as 50 ng. The capillary format of the electronic protein transfer technique allows direct deposition of extracted proteins onto a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) target. Various matrices and solvent compositions are evaluated for the analysis of extracted and concentrated SDS-protein complexes using MALDI-MS. The electronic protein transfer technique, when operated under optimized conditions, is demonstrated for the effective (>70% recovery), speedy (less than 5 min), and sensitive MS identification of gel resolved proteins (as low as 50 ng).     

A high resolution two-dimensional gel electrophoresis and silver staining protocol demonstrated with nuclear matrix proteins.

An improved two-dimensional gel electrophoresis procedure has been developed utilizing isolated nuclear matrix proteins. The proteins of the cellular nuclear matrix are tissue specific. They are an example of a protein set whose two-dimensional electrophoretic patterns afford much information of clinical significance. However, current two-dimensional gel techniques were not completely satisfactory for the small amounts of protein present in tissue samples. There was a need for a two-dimensional gel procedure which was capable of increased sensitivity and resolution and at the same time was reliable and reproducible. This has been accomplished by implementing several modifications to the current two-dimensional gel procedures. In addition, changes were introduced in the silver staining process of the gels to increase the signal to background ratio. The overall procedure affects a dramatic increase in the resolution and clarity of the proteins visualized on two-dimensional gels and is no more laborious than current techniques.     

High performance capillary electrophoresis of calmodulin

The electrophoretic properties of purified calmodulin were investigated. High performance capillary electrophoresis of this Ca2+(-)binding protein in free solution at pH 2.5 resulted in an elution of a single peak with a retention time of approximately 4.7 min. Addition of [ethylene-bis(oxyethylenenitrilo]) N,N,N',N'-tetraacetic acid (EGTA) to the protein prior to capillary electrophoresis completely abolished this electrophoretic profile. Polyacrylamide gel electrophoresis of calmodulin under denaturing and nondenaturing conditions also revealed a single polypeptide band. However, the relative electrophoretic mobilities of this protein could vary, depending on the presence or absence of Ca2+. The pI of calmodulin was estimated to be 3.7 by using isoelectric focusing techniques. Analysis of this acidic protein by high performance capillary electrophoresis at pH 8.0 revealed that it could be resolved into two major and one minor polypeptide peaks, regardless of the presence or absence of Ca2+. These findings suggest that capillary electrophoresis at near physiological pH may differentiate the microheterogeneity of calmodulin.